Rc Gene Research Articles

Yeast has a true stringent response

IN bacteria there is a coordinate regulation of the synthesis of ribosomal RNA and ribosomal protein1. This is most evident from studies of the rates of synthesis of ribosomal components as a function of the growth rate of the cell2,3. Another example is the ‘stringent response’, in which cells deprived of an essential amino acid shut down the transcription of ribosomal RNA as well as that of messenger RNA for ribosomal proteins4,5, but increase the synthesis of mRNA for certain biosynthetic proteins6. Although this stringent response is at least partially due to the rc gene product and/or its synthesis of ppGpp, little is known about the detailed mechanisms involved in controlling the two types of transcription. To what degree does the stringent response exist in eukaryotic cells, which use separate polymerases for the transcription of ribosomal RNA and messenger RNA6? We7 and others8 have shown that in Saccharomyces cerevisiae deprived of an amino acid, the transcription of ribosomal RNA is severely inhibited. Ribosomal RNA transcription is also inhibited in cells treated with cycloheximide7,9, but it continues almost normally in cells in which synthesis of ribosomal proteins has specifically been inhibited through a temperature-sensitive mutation10. We demonstrate here that in cells deprived of an amino acid there is a specific depletion of messenger RNA coding for ribosomal proteins.

Defective translation in RC - cells.

Mutation in the RC gene results in the formation of a species of β galactosidase with reduced specific activity and increased thermolability.

Effect of the RC gene product on constitutive enzyme synthesis

An RC − strain of Escherichia coli which is constitutive for alkaline phosphatase synthesis synthesizes fivefold less alkaline phosphatase during amino-acid starvation than does an isogenic RC + strain. This difference in alkaline phosphatase synthesis is independent of repression.

PpGpp Formation in Escherichia coli Treated with Rifampicin

Abstract It has been previously reported that the accumulation of the regulatory nucleotide ppGpp (MS I), which is governed by the RC gene of Escherichia coli, does not occur in the presence of rifampicin. Since this drug inhibits RNA chain initiation, it was suggested that the formation of ppGpp is somehow dependent on the presence of nascent RNA chains (Wong, J. T.-F., and Nazar, R. N., J. Biol. Chem., 245, 4591 (1970)). We find that this effect of rifampicin depends on amino acid activation: when valyl-tRNA synthetase is specifically inactivated, then ppGpp accumulation proceeds in rifampicin despite complete inhibition of RNA synthesis. The effect of rifampicin on ppGpp formation is therefore indirect, and probably reflects the recharging of transfer RNA due to the fact that the rate of protein synthesis drops in rifampicin-treated cells.

The Mechanism of Amino Acid Control of Guanylate and Adenylate Biosynthesis

Abstract The biosynthesis of GTP and ATP is inhibited during amino acid starvation of RC+ cells. This inhibition is a specific consequence of RC function and cannot be accounted for on the basis of feedback inhibition. The pattern of incorporation of radioactive adenosine into intracellular purine nucleotides suggests that the blocks imposed by the RC function lie before GMP and AMP. The regulatory nucleotide ppGpp (originally designated MS I), whose rapid accumulation in response to amino acid starvation is governed by the RC gene product, inhibits IMP dehydrogenase and adenylosuccinate synthetase, which are the first enzymes of the guanylate and adenylate pathways.

Control of stable-RNA synthesis and maturation of 5-S RNA in Salmonella typhimurium RC str and RC rel

1. Synthesis of stable RNA in Salmonella typhimurium RC str and RC rel during amino acid starvation was studied with the aid of methylated albumin kieselguhr chromatography and polyacrylamide gel electrophoresis. 2. Synthesis of all classes of stable RNA was coordinately controlled by the RC gene under these conditions. The effect of the RC gene therefore appears to be the same in Escherichia coli and S. typhimurium. 3. Amino acid starvation did not result in the accumulation of an abnormal form of 5-S RNA, as in E. coli. However, addition of chloramphenicol, to either starved or exponentially growing cells, did result in the accumulation of such an abnormal form, with properties very similar to those of the E. coli 5-S RNA precursor. 4. Experiments with various low doses of chloramphenicol, indicate that maturation of 5-S RNA is dependent on a minimum rate of protein synthesis, which, under our conditions, lies between 40% and 60% of the rate shown by exponentially growing cells.

Consequences of Expression of the "Relaxed" Genotype of the RC Gene: LIPID SYNTHESIS

Abstract Two pairs of strains that differ in the rel-1 locus were compared for their ability to synthesize protein, RNA, and lipid in the presence and absence of a required amino acid. In the absence of a required amino acid, protein synthesis ceased in both relaxed control (RCrel) and stringent control (RCstr) cells, and RNA synthesis ceased only in the RCstr cells. Lipid synthesis continued at an approximately equal rate in the presence or absence of a required amino acid in both RCrel and RCstr cells. Analysis of the lipid extracts showed a decrease in cardiolipin in RCrel cells deprived of a required amino acid. Otherwise, the distribution of the lipids was not affected by the absence of a required amino acid in either the RCrel or RCstr cells and was quite similar in both types of cells. The fatty acid distribution also appeared unaffected. These results are consistent with the view that the control of RNA synthesis exercised by the rel-1 locus cannot be simply at the level of nucleoside triphosphate availability.

RC gene control in Escherichia coli is not restricted to RNA synthesis

Auxotrophic stringent and relaxed cells of Escherichia coli were incubated in a synthetic medium containing [ 14C]glucose as a sole carbon source. In the absence of the required amino acids, the rate of incorporation of 14C radioactivity into acid-soluble nucleotides, lipids and RNA was found to be greatly reduced in stringent cells. The addition of chloramphenicol restored the incorporation to the same level as the control culture containing both required amino acids and chloramphenicol. On the other hand, in relaxed cells the incorporation of radioactivity was less affected by the removal of amino acids, and chloramphenicol did not show any stimulation. The net increase in amount of various cellular constituents was determined under similar conditions with nonlabeled glucose. The results indicated that there was more pronounced inhibition of accumulation of RNA, DNA, lipids, hexosamines and carbohydrates by starvation of amino acids in stringent cells than in relaxed cells. When chloramphenicol was added, there was always a tendency to recover to some extent from the inhibition in stringent cells but not in relaxed cells. Essentially the same results were obtained in an experiment in which the rate of respiration was studied with glucose as substrate. All these findings suggest that not only RNA synthesis but also many other metabolisms in E. coli cells are under the control of RC gene.

The mechanism of early inhibition by an RNA phage of protein and RNA synthesis in infected cells

R17 infection causes a rapid reduction in the overall rates of both protein and RNA synthesis in host Escherichia coli Hfr 1 cells. The results of the present investigation have led to the conclusions that these early inhibitions are due to a greatly reduced capacity of the infected cells to transport exogenous amino acids. The intracellular functioning of the viral genome is not required for these inhibitions. The following observations support these conclusions: (1) the inhibitions are caused by the UV-inactivated phage as well as by the intact phage; (2) the complete penetration of viral RNA into the host cytoplasm is not required for the inhibition of protein synthesis; (3) the inhibition of amino acid transport can occur even in the presence of chloramphenicol; (4) the viral inhibition of RNA synthesis greatly depends on the allelic state of the RC gene of a host. The inhibition is greater in a stringent than in a relaxed host; (5) the inhibition of RNA synthesis occurs only when the host cells are dependent on the extracellular supply of amino acids. No inhibition of RNA synthesis occurs in the host grown in an amino acid-free medium. The greater inhibition of protein and RNA synthesis occurs at the higher multiplicity. Since phage can grow when there is no inhibition of host macromolecular synthesis, the observed inhibition may play no role in normal infection.

Two compounds implicated in the function of the RC gene of Escherichia coli.

Autoradiography has revealed in a stringent strain of E. coli two compounds that seem to be involved in the inhibition of the synthesis of RNA.

Amino acid-dependent control of the transport of α-methyl glucoside in [formula omitted

RNA synthesis in E. coli is dependent on the presence of a full complement of amino acids and the locus “RC” on its chromosome is responsible for the control of RNA synthesis ( Borek, Ryan and Rockenbach, 1955; Pardee and Prestidge, 1956; Stent and Brenner, 1961). However, we have recently demonstrated that the incorporation of 14C-acetate into lipid fraction is subject to amino acid control in stringent strains of E. coli (Sokawa, Nakao and Kaziro, 1968). Moreover, the synthesis of carbohydrate was shown to be also dependent on the presence of amino acids (Sokawa, Nakao and Kaziro, unpublished). Consequently, it was suggested that the control by RC gene is not limited to RNA synthesis, and many other biosynthetic reactions might be regulated by RC gene. In this paper we wish to report our recent observation that the transport of α-methyl D-glucoside (α-MG), a non-metabolizable analog of glucose permeation system (Kepes and Cohen, 1962), is also under amino acid control in E. coli . The stringent strains fail to accumulate 14C-α-MG during amino acid starvation whereas the relaxed strains continue to uptake 14C-α-MG in the absence of required amino acids. As in the case of RNA and lipid synthesis, addition of chloramphenicol (CM) restores the transport of α-MG under conditions of amino acid starvation in stringent strains.

On the nature of the control by RC gene in [formula omitted]: Amino acid-dependent control of lipid synthesis

On the nature of the control by RC gene in [formula omitted]: Amino acid-dependent control of lipid synthesis

PIGMENTED FAT CELLS IN A MUTANT OF DROSOPHILA MELANOGASTER

1. Cytoplasmic pigment granules are found in some of the fat cells of the recessive mutant, rc, of Drosophila melanogaster. These scattered red fat cells are located chiefly in the thorax and head, but a few occur in the abdomen and appendages of the adult. Using genetic methods it had been shown previously that these pigment accumulations are related to the synthesis of the brown eye pigment of this insect (Jones and Lewis, 1957).2. The pigmentation in the rc fat cells is suppressed when the rc gene is combined with the recessive factor, tuw. This combination, however, in no way alters the expression of the characteristic pattern of tuW as revealed by the presence of melanotic tumors in the caudal fat masses of the homozygous tuwrc flies. After a period of larval starvation, the tuwrc flies develop both the red-pigmented fat cells and melanotic tumors. The time of appearance of the rc pigment has been shifted under these nutritional and genetic conditions. The cytoplasmic pigment granules appear in the cells originating from the anteriormost section of the larval fatbody which is closely associated with the anterior Malpighian tubules. During the reorganization accompanying metamorphosis from the larval to the adult stage, these cells are redistributed mostly to the thoracic and cephalic regions while a few are found in the abdomen and appendages. An explanation is thus provided for the cytodifferentiation of pigmented and nonpigmented fat cells found side by side in the adult fly.3. The nature of the pigment granules has been examined in in vitro preparations at each of these periods of development, and of particular interest is the internal threadlike structure of these cytoplasmic inclusions during the early stages of pigment formation.